Assays for preimplantation factor and preimplantation factor peptides

ABSTRACT

The present invention relates to assay methods used for detecting the presence of PIF, and to PIF peptides identified using this assay. In particular, the present invention relates to flow cytometry assays for detecting PIF. It is based, at least in part, on the observation that flow cytometry using fluorescently labeled anti-lymphocyte and anti-platelet antibodies demonstrated an increase in rosette formation in the presence of PIF. It is further based on the observation that flow cytometry demonstrated that monoclonal antibody binding to CD2 decreased in the presence of PIF. The present invention further relates to PIF peptides which, when added to Jurkat cell cultures, have been observed to either (i) decrease binding of anti-CD2 antibody to Jurkat cells; (ii) increase expression of CD2 in Jurkat cells; or (iii) decrease Jurkat cell viability. In additional embodiments, the present invention provides for ELISA assays which detect PIF by determining the effect of a test sample on the binding of anti-CD2 antibody to a CD2 substrate.

This application claims priority to and is a divisional application ofU.S. Non-provisional application Ser. No. 10/482,244 filed Aug. 11,2004, which is a US National Stage Application under 35 U.S.C. §371claiming priority to PCT/US2002/20599 filed Jun. 28, 2002, which claimspriority to U.S. Provisional Application No. 60/302,607 filed on Jul. 2,2001.

INTRODUCTION

The present invention relates to preimplantation factor (“PIF”) a veryearly marker of fertilization and embryo viability, to new methods fordetecting PIF activity and to PIF peptides.

BACKGROUND OF THE INVENTION

Infertility is a major health care concern affecting millions of couplesworldwide. Contributing to this problem, early demise of the humanconceptus is a common event. Approximately 73% of natural singleconceptions are lost before reaching week 6 of gestation (Boklage C E.Survival probability of human conceptions from fertilization to term.Int J Fertil 1990; 35:75). This is mostly due to early embryonic demiseprior to implantation or soon after implantation occurs. Data relatingto the low fertility rate observed in older women and its improvement byoocyte donation from young women indicate that oocyte quality is animportant factor in achieving a successful pregnancy (Navot D, Bergh PA, Williams M A et al. Poor oocyte quality rather than implantationfailure as a cause of age-related decline female fertility. Lancet 1991;337:1375).

In vitro fertilization (“IVF”) is a technology which has been developedto address the problem of infertility. However, maintaining embryoviability is even more problematic under the artificial conditions usedfor culturing embryos in vitro for implantation. In vitro, the embryodevelopment rate is lower than in vivo and only 25-65% of embryostypically develop to the blastocyst stage (Gardner D K, Lane M,KOuridakis K, Schoolvcraft W B. Complex physiologically based serum-freeculture media increase mammalian embryo development. In: Gomel V, LeungP C K, eds. In vitro fertilization and assisted reproduction. Procc 10thWorld Congress, 1997: 187). The state of the art is not yet able toidentifying embryos likely to implant and survive. Human chorionicgonadotrophin (“hCG”), the currently used marker for fertilization invivo and early embryo implantation, can only be detected several daysafter implantation. As a result of the lack of a suitable marker forembryo viability, nowadays many embryos incapable of implanting arebeing transferred, thus lowering the chance for achieving successfulpregnancy.

To address the possibility that embryos may not be viable, a greaternumber of embryos are simultaneously transferred into a potentialmother. The transfer of a high number of embryos may lead to multiplepregnancies, which are inherently risky, while transfer of a smallnumber of embryos carries the risk that none would implant, losing awhole IVF cycle. Clearly, there is a need to improve embryo selectionand define accurate markers to determine embryo viability. In addition,using non-invasive methods by testing culture media for productsspecific to viable, implantation-competent embryos would allow selectionof those most likely to result in successful pregnancies, withoutcausing embryo damage.

Another factor involved in determining whether a pregnancy is successfulor not is the interaction between the conceptus and the mother's immunesystem. Shortly after fertilization a systemic maternal recognition ofpregnancy should occur. The mother's immune system modulation triggeredby specific early embryo signals could be the key of this process. Oncethe oocyte is fertilized, the zygote up to hatching blastocyst issurrounded by the zona pellucida, a hard semi permeable membrane.Therefore the embryo-maternal communication must occur simultaneouslywhile the embryo is developing in the oviduct and uterine cavity throughcompounds that are secreted by the embryo.

It has been shown that pregnant sera and viable embryo conditionedculture media can produce an increase in rosette formation by plateletsand T lymphocytes in the presence of CD2 antibody. As disclosed in U.S.Pat. No. 5,646,003 by Barnea et al., issued Jul. 8, 1997, and in U.S.Pat. No. 5,981,198 by Barnea et al., granted Nov. 9, 1999, the presenceof Preimplantation Factor (“PIF”) can be detected by mixing lymphocytes,platelets, heat inactivated serum from a pregnant subject, guinea pigcomplement, and T11 (anti-CD2) monoclonal antibody (Dakko, Denmark),where rosette formation between platelets and lymphocytes is increasedby PIF in pregnant subjects. PIF has been found to be (i) secreted byviable early human and mouse embryos from the two-cell stage onward;detectable in the peripheral circulation 3-4 days after embryo transferfollowing IVF; (iii) associated with 73% take home babies vs 3% in earlynegative PIF results; (iv) detectable 5-6 days after intrauterineinsemination; (v) absent in non-pregnant serum, or non-viable embryos;and (vi) present in various pregnant mammals in addition to humans,including mice, horses, cows and pigs. In addition, PIF has beenobserved to disappear from the circulation two weeks before hCGsecretion declines in cases of spontaneous abortion.

The monoclonal antibody used in the above-mentioned PIF assay isdirected toward the lymphocyte associated antigen referred to as CD2.CD2 is present on about 80-90% of human peripheral blood lymphocytes,greater than 95% of thymocytes, all T lymphocytes that form erythrocyterosettes and a subset of NK cells. Various roles for CD2 in T cellactivation have been proposed, including function as an adhesionmolecule which reduces the amount of antigen required for T cellactivation and as a costimulatory molecule or direct promoter of T cellactivation. Moreover, CD2 has been implicated in the induction ofanergy, the modulation of cytokine production and the regulation ofpositive selection of T-cells.

The natural ligand for CD2 is the structurally related IgSF CAMs CD58(LFA-3), a cell-surface adhesive ligand with broad tissue distribution.In addition, CD2 can interact with CD48, CD59 and CD15 (Lewisx)-associated carbohydrate structure. CD2 binds CD58 with very lowaffinity and an extremely fast dissociation constant The lateralredistribution of CD2 and its ligand CD58 also affect cellular adhesionstrength. Regulation of CD2 adhesiveness affects the ability of CD2 toenhance antigen responsiveness. CD2-cell lines incapable of avidityregulation exhibit a marked deficiency in an antigen-specific response.Strength of adhesion resulting from increased CD2 avidity contributesdirectly to T-cell responsiveness independently of CD2-mediated signaltransduction.

SUMMARY OF THE INVENTION

The present invention relates to assay methods used for detecting thepresence of PIF, and to PIF peptides identified using this assay. Inparticular, the present invention relates to flow cytometry assays fordetecting PIF. It is based, at least in part, on the observation thatflow cytometry using fluorescently labeled anti-lymphocyte andanti-platelet antibodies demonstrated an increase in rosette formationin the presence of PIF. It is further based on the observation that flowcytometry demonstrated that monoclonal antibody binding to CD2 decreasedin the presence of PIF.

The present invention further relates to PIF peptides which, when addedto Jurkat cell cultures, have been observed to either (i) decreasebinding of anti-CD2 antibody to Jurkat cells: (ii) increase expressionof CD2 in Jurkat cells: or (iii) decrease Jurkat cell viability. Inadditional embodiments the present invention provides for ELISA assayswhich detect PIF by determining the effect of a test sample on thebinding of anti-CD2 antibody to a CD2 substrate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-B. PIF purification from mouse embryo culture conditioned medium(MECCM); (A) shows a high performance liquid chromatography (“HPLC”)profile of MECCM-3 kDA ultra-filtrate previously purified by MabCD2affinity chromatography; (B) shows the profile following additional HPLCpurification of a PIF-active fraction from (A).

FIG. 2A-C. Western blot analysis of different PIF peptides purified fromMECCM; MabCD2 was used as a primary antibody and anti-sense mousehorseradish peroxidase (HRP)-biotin streptavidin complex was used assecondary antibody. Specific PIF bands were identified by the ECLdetection reagents (Amersham Pharmacia Biotech).

FIG. 3A-B. (A) shows flow cytometric determination oflymphocyte-platelet rosette formation (L-P) in the presence of freshculture medium (CM) and mouse embryo culture conditioned medium (MECCM)and MabCD2. Fluorescent labeled specific antibodies to L (MabCD45-PE)and to P (MabCD42a-FITC) were used to detect the L-P complex. MECCM gave3Q-40 percent higher formation of L-P compared to culture medium (CM).(B) shows FC of MECCM effect on MabCD2 binding to Jurkat cells (JC). JCwere incubated with samples and further with MabCD2 Cy5. Antibodybinding to CD2 decreased by PIF present in MECCM. Arrows indicate PIFactivity.

FIG. 4A-C. Mass spectrum from PIF peptides purified from MECCM.Molecular weight (MW) of PIF-active fractions from MECCM purified byultrafiltration, diafiltration, HPLC, MabCD2-affinity chromatography andby additional HPLC was determined by mass spectroscopy. MW of PIFpeptides were A) 610-995 Da; B) 963-1848 Da; and C) 1807-1846 Da.

FIG. 5A-F. Flow cytometric analysis of PIF negative effects on MabCD2binding (A), fluorescence (B) and viability (C) in Jurkat cells, and ofPIF positive effects on MabCD2 binding (D), fluorescence (E) andviability (F). PIF in positive samples competes with CD2 (arrowsindicate PIF activity).

FIG. 6. Effect of Synthetic PIF peptides on CD2 expression on Jurkatcells.

DETAILED DESCRIPTION OF THE INVENTION

In a first set of embodiments, the present invention provides for amethod for determining the presence of preimplantation factor in asample, comprising the step of detecting whether the sample contains acomponent which inhibits the binding of an anti-CD2 antibody to CD2antigen; wherein the ability to inhibit the binding of anti-CD2 antibodyto CD2 has a positive correlation with the presence of preimplantationfactor.

Such a method may, for example, be employed in a flow cytometry methodor in an enzyme-linked immunosorbent assay method, using techniquesotherwise known in the art. A non-limiting example of a flow cytometrymethod for detecting anti-CD2 antibody binding to CD2 is presented inSection 7, below.

An anti-CD2 antibody, as that term is used herein, may be a monoclonalor polyclonal antibody which specifically binds to CD2. Such amonoclonal antibody is sold by Pharmigen (see below).

CD2 antigen may be in the form of purified CD2 antigen or may be carriedby a cell. In non-limiting embodiments of the invention, the cell is aJurkat cell. Other CD2-expressing cell lines are known in the art.

The sample may be a serum sample (for example, serum from a subject tobe tested for fertilization/implantation/persistence of embryo), may bea sample of culture fluid (for example, to determine the viability ofembryos prior to transfer for IVF), or may be a solution to be testedfor the presence of a PIF peptide (for example, during the purificationof PIF acting agents; see Section 6, below).

The subject may be a human subject (for example, a human suspected ofbeing pregnant) or a non-human subject (for example an agriculturalanimal or a zoo animal).

In a second set of embodiments the present invention provides for amethod for determining the presence of preimplantation factor in asample, comprising the step of detecting, by flow cytometry, whether thesample contains a component which increases the formation of rosettesbetween lymphocytes, platelets, and anti-CD2 antibodies, where anincrease in rosette formation has a positive correlation with thepresence of preimplantation factor. Such an assay may be performed, forexample, using fluorescently labeled antibodies directed towardlymphocytes and platelets, where preferably different labels are usedfor anti-platelet and anti-lymphocyte antibodies.

The increase is relative to a known negative control.

The present invention also provides for the following isolated peptides:

(1) An isolated peptide having a sequence selected from the groupconsisting of: Met-Val-Arg-Ile-Lys-Pro-Gly-Ser-Ala (SEQ ID NO: 1);Met-Val-Arg-Ile-Lys-Pro-Gly-Ser-Ala-Asn-Lys-Pro-Ser (SEQ ID NO: 2);Met-Val-Arg-Ile-Lys-Tyr-Gly-Ser-Tyr-Asn-Lys-Pro-Ser-Asp (SEQ ID NO: 3);and Met-Val-Arg-Ile-Lys-Pro-Gly-Ser-Ala-Asn-Lys-Pro-Ser-Asp-Asp (SEQ IDNO: 4), or an isolated peptide comprising said peptide which binds toanti-CD2 antibody and which is not a circumsporooite protein;

(2) An isolated peptide having a sequenceSer-Gly-Ile-Val-Ile-Tyr-Gln-Tyr-Met-Asp-Asp-Arg-Tyr-Val-Gly-Ser-Asp-Leu(SEQ ID NO: 5), or an isolated peptide comprising said peptide whichbinds to anti-CD2 antibody and which is not an HIV protein;

(3) An isolated peptide having a sequenceVal-Ile-Ile-Ile-Ala-Gln-Tyr-Met-Asp (SEQ ID NO: 6 or an isolated peptidecomprising said peptide which binds to anti-CD2 antibody; and

(4) An isolated peptide having a sequence selected from the groupconsisting of Ser-Gin-Ala-Val-Gln-Glu-His-Ala-Ser-Thr (SEQ ID NO: 7) andSer-Gln-Ala-Val-Gln-Glu-His-Ala-Ser-Thr-Asn-Xaa-Gly (SEQ ID NO: 8),where Xaa can be any amino acid, or an isolated peptide comprising saidpeptide which binds to anti-CD2 antibody and which is not a silencingmediator for human retinoid and thyroid hormone.

EXAMPLE Identification of PIF Peptides

PIF was isolated from a large volume of MECCM using ultra filtration,lyophilization, high performance chromatography (HPLC), affinitychromatography and western blot. Two-cell-to blastocyst stage mouseembryos were cultured for several days in Ham's F-10 medium withpenicillin, streptomycin, MgSO₄, NaHCO₃, KHCO₃, and Ca lactatesupplemented with 0.1% BSA. MECCM collected was stored at −80° C. untilused.

One liter of MECCM was purified by ultra filtration through an Amiconmembrane (3 kDa cut-off, YM-3 kDa, Amicon. Millipore Co, USA).Concentrated MECCM was further diafiltered using 300 ml of pure water.In addition, fresh culture media (CM, without embryos) was processed inthe same way. Only MECCM-3 kDa ultra filtrate and diafiltrateddemonstrated PIF activity and then they were pooled and concentrated bylyophilization.

It was observed that PIF is able to bind to anti-CD2 monoclonal antibody(“MabCD2”). Therefore, PIF-active fractions were purified first byaffinity chromatography performed with agarose-hydrazide-MabCD2activated gels. An antibody affinity matrix was prepared as follows. 1.5mg of MabCD2 (clone RPA-2.10, Pharmigen, Becton Dickinson) was bufferexchanged with the coupling buffer pH 5.5 using the Econo-Pac 10DGdesalting column provided and further oxidized with sodium periodate andcoupled to 2 ml of agarose-hydrazide activated gel following themanufacturer's indications (Affi-gel Hidrazide immunoaffinity kit,BioRad Laboratories, CA, USA). Then, MECCM-3 kDa ultrafiltrate-diafiltrate lyophilized powder was further purified using theMabCD2-affinity chromatography column (10×20 mm). 2 g of MECCM-3kDapowder were dissolved in 10 ml of pure water, pH neutralized, filter-outthrough a 0.22 m syringe sterile filter (Corning Inc., NY, USA) andpassed 5 times through the affinity chromatography column at gravityflow. The column was washed-out with 5 volume bed of 100 mM phosphatesaline buffer, pH 7.2, followed by washing with 5 volume bed of 0.5 MNaCl.

The bound PIF was eluted with 3 ml of 0.1 M acetic acid. PIF-elutedfractions were pooled, assayed for PIF activity and concentrated bylyophilization.

A total of 300 mg of MECCM-3 kDa ultra filtrate further purified byaffinity chromatography were run in three batches by HPLC on a ClipeusC18 preparative column (Higgins Analytical, Inc., USA). Preparative HPLCrunning parameters were: flow, 15 ml/min. Buffers: A=0.1%trifluoroacetic acid (TFA); B=0.1% TFA in 99.9% acetonitrile (CH3CN).Gradient: 0% B, during 5 min plus 0-60% B for 30 min and 0-100% B for 3min.

Fractions from HPLC were further concentrated by evaporation. HPLCconcentrated fractions were pH neutralized and re-assayed forPIF-activity. Several fractions showed high PIF-activity (see FIG. 1A).These fractions were purified by additional HPLC on a Vydac C8analytical column (4.6×250 mm; Hesperia, Calif., USA). Additional HPLCrunning parameters: flow, 1 ml/min. Buffers: A=0.1% trifluoroacetic acid(TFA); B=0.1% TFA in 99.9% acetonitrile (CH3CN). Gradient: 0% B, during5 min plus 0-60% B for 30 min and 0-100% B for 3 min. Several elutedfractions showed PIF activity (see FIG. 1B) and were further sequencedfor amino acid composition and their molecular weight (MW) wasdetermined by mass-spectrometry.

PIF active fractions purified from MECCM gave positive signals inWestern blots (“WB”). Solutions from CM ultra filtrate-lyophilizefraction was used as negative control in WB. The WB conditions were asfollows. For gels, SDS-PAGE pre-casting gels (BioRad) were used, havinga 16.5% agarose resolving gel and a 4% agarose stacking gel. The gelswere run in 100 mM Tris, 100 mM Tricine, 0.1% SDS, pH 8.3 (Tris-tricinerunning buffer). Samples consisting of 30 microliters of PIF-MECCMpurified fractions plus 10 microliters of Tricine sample buffer [200 mMTris (hydroxymethyl)aminomethane (Tris-HCl) pH 6.8, 2% sodium duodecylsulphate (SDS), 40% glycerol, 0.04% Coomassie blue brilliant (CBB-G250)](BioRad) were incubated at 95° C. during 5 min. After cooling, thesamples were loaded into the wells of the SDS-polyacrylamide gels(PAGE). To determine the molecular weight of low molecular weight (MW)polypeptides, 10 microliters of a 1:20 water dilution of SDS-PAGEstandards (BioRad) were loaded into a well of each gel. Forelectrophoresis, samples and standards were run at 175 v during 5 minplus 60 v for 1 h.

The resulting gels were then electro-blotted using, as transfer buffer,100 mM CAPS [3-(cyclohexylamino)-1-propanesulfonic acid) buffer, pH 11.Electro-blotting was performed at 80 mA during 1 h onto a 0.22 μmnitrocellulose membrane (BioRad).

Then, nitrocellulose membranes were blocked with 5% blocking solutions(Amersham. Pharmacia, Biotech, NJ, USA) at room temperature during 18h., and then were washed-out 4 times during 20 min with PBS-T [phosphatesaline buffer −0.05% polyoxyethylenesorbitan monolaurate (Tween 20)].For the primary antibody incubation, blocked membranes were incubatedwith 2 μg/ml MabCD2 (Pharmigen)—PBS-T solutions at room temperatureduring 2 h, and then washed as above. For the secondary antibodyincubation, the membranes were incubated with anti mouse IgG-horseradish peroxidase conjugate (1:1000 in PBS-T) solution at roomtemperature during 1 h. PIF bands were then visualized using theECL-chemiluminescent system (Amersham). FIG. 2 shows a typical WB of PIFpeptides purified from MECCM.

A flow cytometric methodology (FC) for measuring PIF was developed toimprove the efficiency and reproducibility of methods set forth in U.S.Pat. Nos. 5,646,003 and 5,981,198. In particular, rosette formation wasevaluated by FC with pregnant and non-pregnant human and porcine serum,MECCM, CM and isolated PIF-fractions using MabCD2 or MabCD2-Cy5(Cy-chrome conjugated antibody), MabCD45-PE (phycoerytrhin conjugatedantibody) and MabCD41a-FITC (fluorescein isothiocyanate conjugatedantibody), all antibodies were from Pharmigen. The ratio of labeled P-Lcomplex was higher by 30-40% with MECCM versus CM (FIG. 3A). Further, itwas found that pre-incubation of MECCM or pregnant sera with immobilizedMabCD2 prevented the P-L formation in the assay. The addition of aMabCD58 (lymphocyte function-associate antigen-3 or LFA-3) antibody toL-P did not prevent totally the rosette formation by effect ofPIF-active samples in the assay.

A FC-PIF quantitative assay using Jurkat cells (JC) and MabCD2-Cy5 wasdeveloped (FIG. 3B). The use of an immortalized leukemia cell lineavoids the need for fresh donor blood to assess the PIF activity by thebioassay. The JC-FC assay was validated with human serum samples (seeTable I) and was used to assess PIF activity of fractions during PIFpurification.

MW of purified PIF-active fractions was determined by mass spectralanalysis on a Voyager-RP Biospectrometry MALDI-TOF Workstation fromPerseptive Biosystems (Cambrigde, Mass., USA). Samples were mixed with amatrix consisting in a 1:2 mixture of acetonitrile:water containing 1%trifluoroacetic acid. Spectra were averages of approximately 200 scans.PIF-peptides from MECCM have MW between 610-1845 Da (FIG. 4).

Further, it was assessed that pre-incubation of PIF-active fractionswith MabCD2 abolished the PIF-activity. These data indicated that PIFcould be a portion of CD2 or homologue peptides. However, after thesequencing of purified PIF peptides it was demonstrated that thesepeptides are not a portion of CD2 and their amino acid sequences areunique.

Using the JC-FC assay it was demonstrated that MEECM-PIF peptides havethree different effects on CD2 expressed by T cells. These effects arerelated to:

decreasing MabCD2 binding to the JC; up-regulating CD2 expression by JC;or decreasing JC viability.

Purified PIF active fractions from mouse embryos were sequenced by Edmandegradation on an Applied Biosystems Pulsed Liquid Sequencer (model477A). Released amino acids were derivatized with phenylisothiocyanateto give the PTH-amino acids which were detected by reverse phase-HPLC ona HPLC system in line with the sequencer. Several of the PIF fractionsyielded unique sequences. Several peptides gave sequences whoseN-terminal nine and ten residues were identical indicating that thepeptides were various truncated forms of common molecules (see TableII). PIE peptides were identified as a least three unique families ofembryo-derived and pregnancy-related small peptides. The amino acidsequence of a family of three PIF peptides matches 100% with a region ofCircumsporozoite protein (malaria parasite: Plasmodium falciparum) (SEQID Nos. 1, 2 and 4). This family of PIE peptides up regulates the CD2expression by JC. A PIF peptide (14 amino acids) (SEQ ID No. 3) thatshares only the five first amino acid residues with the former describedPIF-peptide's family and another PIE peptide (18 amino acids) (SEQ IDNo. 5) that matches in 11 amino acids to the sequence of HIV-1 RNAdirected DNA polymerase (reverse transcriptase, EC 2.7.7.49) also upregulate the CD2 expression by JC. In addition, another family of twoPIT peptides (10 and 13 amino acids) (SEQ ID No. 7 and 8) matches in 10amino acids with the sequence of the human receptor-interacting factor,a silencing mediator for retinoid and thyroid hormone receptor (SMRT)(Chen and Evans, 1995). The shorter member of this PIE-peptide familyshows a competitive effect for the binding of MabCD2 to JC and thelonger PIF-peptide decrease the viability of JC. It is worth to noticethat transcriptional silencing mediated by nuclear receptors isimportant in development, differentiation and oncogenesis.

PIF peptides were synthesized by solid-phase peptide synthesis (SPPS) onan Applied Biosystems Peptide Synthesizer employing Fmoc(9-fluorenylmethoxycarbonyl) chemistry in which the amino nitrogen ofeach amino acid is blocked with Fmoc. Coupling was performed byactivation of the carboxyl groups of the N-protected amino acids using 3mmol/ml of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate/1-hydroxybenzotriazole on the presence ofdiisopropylethylamine. Activated amino acids were sequentially added tothe nascent peptide. Upon completion of the synthesis, finalpurification was carried out by reversed-phase HPLC and identity wasverified by MALDI-TOF mass spectrometry and amino acid analysis. PIFsynthetic peptides demonstrated to have similar effect on CD2 phenomenonin Jurkat cells (FIG. 6), and were also immunodetected by the Mab CD2.

Flow Cytometry Assay for PIF Materials

Materials included Jurkat leukemia cells (JC); cloning medium; Falcontubes for flow cytometry measurements; Mab CD2-Cy5 (Cy-chrome conjugatedantibody, clone RPA-2.10, Pharmigen, Becton Dickinson); the biologicalsample (which could be a human serum to be assayed for PIF activity, orcould be a solution of a putative or synthetic PIF peptide); PBS-2% BSA(100 mM phosphate saline buffer −2% bovine serum albumin; a negativecontrol); trypan-blue dye; a CO₂-incubator for cell culture; and a flowcytometer.

Method

To Prepare the JC Suspension:

Check the viability of the JC culture using Trypan blue dye exclusionstaining. Cell viability should be between 80-90%.

Wash twice the JC with 10 ml of PBS-2% BSA.

Prepare a JC suspension in cloning medium or PBS-2% BSA containing5,000,000 cells/ml.

Dispense 50 ml of JC suspension into falcon tubes (250,000 cells/tube).

For the Sample Incubation:

Add 200 ul samples, serum from early pregnancy controls (3 positivecontrols) or PBS-2% BSA (negative control). Mix gently.

Incubate at room temperature for 20-30 min.

Add 200 ul of MabCD2-Cy5 diluted 1:200 in PBS-2% BSA. Mix gently.

Incubate at room temperature for 20-30 min.

For Flow Cytometric Determination:

Measure the fluorescence of each tube (488 nm laser excitationwavelength). Compare the fluorescence of alive and total cells and totaldead cells (see FIG. 5) with controls.

Calculate PIF Activity as Follows:

Fluorescence of total cells/% dead cells×Fluorescence of alive cells

Interpretation of the Results

PIF negative activity should be in the range of: 130-340

Positive PIF samples are out side of the negative reference range

Various publications are cited herein, the contents of which are herebyincorporated by reference in their entireties.

TABLE I CLINICAL VALIDATION OF THE FLOW CYTOMETRIC PIF ASSAY IN SERUMFROM 37 PATIENTS Positive Negative predictive predictive SensitivitySpecificity Value Value Samples (%) (%) (%) (%) Early 91 92 92 92pregnancy Mid 66 92 86 73 pregnancy Late 100 92 83 92 Pregnancy Allpregnant 86 92 88 86 Samples

TABLE II Amino acid sequence of PIF peptides derived frompre-implantation mouse embryos PEPTIDE CODE/ T CELL AMINO RELATEDSEQUENCE ACID CD2 MATCHING NUMBER SEQUENCE ACTIVITY (REFERENCE) MECCM-4[H]-m-v-r-i-k-p- Increase Match 100% in 9 (9 AA) g-s-a[OH] of CD2 AAwith (SEQ ID NO: 1) expression circumsporooite protein (malariaparasite- Plasmodium falciparus) MECCM-6 [H]-m-v-r-i-k-p- Increase Match100% in 9 (13 AA) g-s-a-n-k-p-s- of CD2 AA with circum- [OH] expressionsporooite protein (SEQ ID NO: 2) (malaria parasite- Plasmodiumfalciparus) MECCM-3 [H]-m-v-r-i-k-p- Increase Match 100% in 9 (15 AA)g-s-a-n-k-p-s-d- of CD2 AA with circum- d-[OH] expression sporooiteprotein (SEQ ID NO. 4) (malaria parasite- Plasmodium falciparus) MECCM-5[H]-m-v-r-i-k-y- Increase Match 100% in (14 AA) g-s-y-n-k-p-s-d- of CD2SAA with circum- [OH] expression nsporooite protein (SEQ ID NO: 3)(malaria parasite- Plasmodium falciparus) MECCM-7 [H]-s-g-i-v-i-y-Increase Match 100% (18 AA) q-y-m-d-d-r-y-v- of CD2 in 11 AA withg-s-d-l-OH] expression HIV-RNA- (SEQ ID NO. 5) DNA directed polymerase(EC 2.7.7.49) P 14-6 [H]-v-i-i-i-a-q-y- Competition No match (9 AA)m-d-[OH] with (SEQ ID NO: 6) MabCD2 for binding P13-5 [H]-s-q-a-v-q-e-Decrease Match 100% (10 AA) h-a-s-t-OH] of cell in 10 AA with (SEQ IDNO: 7) viability a silencing mediator for human retinoid and thyroidhormone receptor (SMRT) P4-3 [H]-s-q-a-v-q-e- Decrease Match 100% in (13AA) h-a-s-t-n-a-g- of cell 10 AA with a OH] viability silencing mediator(SEQ ID NO: 8) for human retinoid and thyroid hormone receptor (SMRT)

1. An isolated peptide which binds to anti-CD2 antibody, said isolatedpeptide comprising the sequenceSer-Gly-Ile-Val-Ile-Tyr-Gln-Tyr-Met-Asp-Asp-Arg-Tyr-Val-Gly-Ser-Asp-Leu(SEQ ID No: 5).